ORAC抗氧化.doc

96孔板ORAC抗氧化快速监测方案参考文献：Tao Wu, Jun Yan, Ronghua Liu, Massimo F. Marcone, Haji Akber Aisa, Rong Tsao,Optimization of microwave-assisted extraction of phenolics from potato and its downstream waste using orthogonal array design,Food Chemistry,2012 ,133 (4) :1292-1298Reagents 1) Phosphate buffer (75 mM, pH 7.4): NaH2PO4 (Mw119.98): 1.71 gNa2HPO4 (Mw.141.96): 8.62 gDissolve in 900 mL dH2O, adjust pH to 7.4 using HCl or NaOH. Add more dH2O to final volume 1L2) Fluorescein stock solution (144.6510-3 mM): weigh 4.8mg of fluorescein in a 2 mL mirofuge tube, dissolve in 1 mL MeOH and transfer into a 100 mL volumetric flask. Rinse the tube 3 times with 1 mL MeOH and quantitatively transfer into the volumetric flask. Add 75 mM phosphate buffer to 100 mL mark. Wrap with foil and store at 5 oC (good to 6 months).Fluorescein working solution (8.6810-5mM): Add 9 mL of stock to 14.991 mL of 75mM phosphate buffer (pH 7.4). The diluted solution was made fresh daily.3) Trolox standard stock solution (0.02M): 25 mg of Trolox is dissolved in 5 mL MeOH.Trolox working solution: 50 mL of stock solution was diluted with 9.95ml of 75mM phosphate buffer (pH 7.4). Final concentration was 100M.4) AAPH solution: 0.414g of AAPH was dissolved in 10ml of 75mM phosphate buffer (pH 7.4) to a final concentration of 153mM and made fresh daily. 5) Sample preparation: dilute the samples with phosphate buffer (you will need to try to find out the appropriate dilutions for your samples, could be 10-, 100- and 1000-fold). If samples are not soluble in buffer, use the ORAC-Lipophilic protocol.Procedure:1) Blank wells received 25 l of buffer .2) Standard wells received 25 l of Trolox dilution (6.25, 12.5, 25.0, 50.0, 100M).3) Sample wells received 25 l of appropriately diluted samples.4) 150l of 8.6810-5mM fluorescein working solution was added into all experimental wells5) The plate was allowed to equilibrate by incubating for a minimum of 30 min in the Synergy HT Multi-Detection Microplate Reader at 37C. 6) Reactions were initiated by the addition of 25 l of AAPH reagent followed by shaking at maximum intensity for 10 seconds. The fluorescence was then mointored kinetically with data taken every minute.Operation of a Synergy HT Multi-Detection Microplate Reader 1. Open windows and turn on instrument. Click Gen5, create a protocol.1) Define the procedure: Temperature 37C, start kinetic ( run time 60 min, interval 1min, Read: em 485/20, ex 528/20, Delay 1530min, Shake 10sec )2) Plate layout: Type, ID Prefix, Conc.3) Data Reduction: Gen 5 automatically creates two steps: the blank subtraction transformation and well analysis for MaxV.Cllick Well Analysis Enter a label e.g. AUC (NET AUC) Select Formula and enter (R1/R1)+(R2/R1)+(R3/R1)+.+(R61/R1) Click OK Click Transformation Enter a label e.g. AUC or (NET AUC) Select Formula and enter X or (X-BLK) Click OK Click Curve Analysis Data In: X-Axis Data (Plate Layout Settings). Y-Axis Data (AUC or NET AUC Formula Result 485/20, 528/20 ) Click OK Save the protocol Click protocol, select a protocol. You can run it in an experiment2. Click preheat , Select on3. When the temperature reach to 37 C, put the plate in and click read, enter plate name. 4. After reading, save the experiment name. Click plate name Matrix: layout, data, kinetic curveGraph: standard curve, sample curve,Statistics: Results AUC, NET AUC, TROLOX Concentration. Calculated the sample slop is Trolox equivalent concentration. The ORAC procedure used an automated plate reader(KC4, Bio Tek, USA) with 96-well plates (Prior et al.,2003). Analyses were conducted in phosphate buffer pH 7.4at 37 1C. Peroxyl radical was generated using 2, 2-azobis(2-amidino-propane) dihydrochloride which was preparedfresh for each run. Fluorescein was used as the substrate.Fluorescence conditions were as follows: excitation at485nm and emission at 520nm. The standard curve waslinear between 0 and 50 mM Trolox. Results are expre